The sugar chain of glycoprotein or the like is known to play a very important role in vivo. Thus, sugar chain engineering, by which the sugar chain structure is intendedly altered, is now an essential technical field. Current techniques for altering sugar chains include, for example, a chemical technique which binds chemically-synthesized target sugar chains to a protein; a biological technique which alters genes for the synthesis of a sugar-chain within a cell by a genetic engineering technique, or produces glycoprotein by altering a host which produces glycoprotein; and a method of sugar-chain synthesis which uses sugar-chain synthetic enzymes.
Progress in the chemical technique is now opening up a way for mass synthesis, however, it is not yet able to easily provide all the types of sugar chains owing to the complexity of sugar chains. On the other hand, thanks to the development of genetic engineering, in the biological technique, control of the expression of genes involving in sugar-chain synthesis is now available, enabling alteration of sugar chains. However, uniform synthesis of all the types of sugar chains is currently difficult, and usually a mixture of different types of sugar chains exists within the product.
In contrast to these techniques, in vitro sugar-chain synthesis using sugar-chain synthetic enzymes is very useful in the synthesis of sugar-chains with a uniform structure. In particular, the combination of such a technique and the biological technique enables mass production of uniform sugar chains.
However, while in vitro sugar-chain synthesis requires the use of sugar nucleotides as sugar donors for glycosyltransferase, the prohibitively high cost of producing sugar nucleotides makes it difficult to apply the method to mass production. That is, sugar nucleotides are present in a very small amount in vivo, and are very reactive, unstable substance which are linked by a high-energy bond. Therefore, only a small amount of sugar nucleotides is produced in each organism, and mass production thereof is difficult.
In recent years, a production system using bacteria has enabled a more practical mass production system of relatively many types of sugar nucleotides, and has enabled a more stable supply of sugar nucleotides. However, a production system with a relatively long reaction process results in low yield, since the system comprises the steps of mixing two types of microorganisms and performing the production using disrupted cells in order to introduce a material contained in cells into other cells. Thus, development of a new technique is being sought.
Among sugar nucleotides, GDP-L-fucose is essential as a sugar donor of fucosyl-transferase for synthesis of sugar chains containing fucose. Sugar chains with the fucose moiety added thereto often play a functionally important role, and therefore providing the sugar donor in large quantities at low cost has been awaited. It has been reported that the GDP-L-fucose is synthesized through 3 reaction steps from GDP-D-mannose, and these 3 reaction steps are catalyzed by two types of enzymes (FIG. 1) (Tonetti et al., J. Biol. Chem., Vol. 271, 27274 (1996)). These enzymes are generally distributed among any organism utilizing fucose, including prokaryotes, such as Escherichia coli, and also eukaryotes, such as higher mammals, for example humans. However, these organisms consume the synthesized GDP-L-fucose, so that GDP-L-fucose does not accumulate within their cells. Accordingly, isolation of GDP-L-fucose from a living organism results in a very small amount of GDP-L-fucose at a high cost. Moreover, synthesis of GDP-L-fucose also requires a long process. Under such present circumstances, it is difficult to supply a sufficient amount of GDP-L-fucose using the above bacterial system.
Two types of the enzymes which catalyze the 3 reaction steps are GDP-D-mannose-4, 6-dehydratase, which catalyzes the first reaction step to convert from GDP-D-mannose to GDP-4-keto-6-deoxy-D-mannose by dehydration reaction; and GDP-4-keto-6-deoxy-D-mannose-3, 5-epimerase-4-reductase which catalyses the subsequent two reaction steps, epimerization and reduction. For the plant Arabidopsis (Arabidopsis thaliana), MUR1 has already been isolated as a gene for GDP-D-mannose-4, 6-dehydratase which catalyzes the first reaction (Bonin et al., Proc. Natl. Acad. Sci. USA, Vol. 94, 2085 (1997)).
However, isolation of a gene for GDP-4-keto-6-deoxy-D-mannose-3, 5-epimerase-4-reductase which catalyzes the subsequent reactions has not been reported. Only a sequence having a high homology with that of the genes for GDP-4-keto-6-deoxy-D-mannose-3, 5-epimerase-4-reductase from another biological species has been submitted to a gene database.